Abstract A key aspect to antiretroviral target identification is understanding the nature molecular interactions, which when perturbed, can result interfere with human immunodeficiency virus type 1 (HIV-1) replication. An underexplored aspect of the HIV-1 replication cycle for discovery of novel antiviral targets has been the steps involved in virus particle assembly. This is due, in part, to the relatively poor understanding of this phase of virus replication ? specifically as it relates to the behavior of Gag movement to the plasma membrane, the engagement of particle budding sites, the molecular Gag-Gag interactions that create the immature Gag lattice, and subsequent particle biogenesis. Detailed comparative analysis of close relatives can be highly informative for the discovery of key antiretroviral targets for drug development. For instance, recent evidence indicates that Gag-Gag interactions differ among retroviruses, which helps explain morphological differences among immature retrovirus particles. Furthermore, we have made key preliminary observations of differences in the pathways for Gag nucleation leading to punctum formation, as well as the nature of particle biogenesis also remain poorly understood aspects of the retrovirus assembly pathway, particularly among human immunodeficiency virus type 1 and its close relatives ? i.e., human immunodeficiency virus type 2 (HIV-2) and human T-cell leukemia virus type 1 (HTLV-1). In this application, we propose to investigate retrovirus immature particle structure and particle biogenesis through innovative state-of-the-art experimental approaches. In particular, we will apply cryo-electron microscopy/tomography (cryo-EM/ET), total internal reflection fluorescence (TIRF) microscopy, photoactivated localization microscopy (PALM), and the novel single-molecule technology of fluorescence fluctuation spectroscopy (FFS) in living cells to investigate 1) comparative analysis of immature Gag lattice structures, 2) investigate the nature of human retrovirus particle biogenesis, and 3) investigate the pathways for the nucleation of Gag oligomerization. These novel studies harness innovative technologies in order to provide new insights into a highly significant and poorly understood aspect of the HIV-1 life cycle and lead to the identification of antiretroviral targets for exploiting by intervention using small molecules.